The Prothrombin Time (PT) test is widely used to monitor oral anticoagulation therapy by coumarins, as a general screening test for the blood clotting system, and as the basis for specific Factor assays. Clotting times obtained with the PT are primarily dependent on the plasma levels of the vitamin K-dependent coagulation Factors II (prothrombin), VII, and X, and on the levels of two non-vitamin K-dependent proteins, Factor V and fibrinogen. Coumarin treatment antagonizes the vitamin K carboxylase/reductase cycle, thus inhibiting the post-translational conversion of glutamate residues to gamma-carboxyglutamate. Vitamin K-dependent clotting factors contain essential gamma-carboxyglutamate residues in their Gla domains. Patients receiving coumarin therapy will therefore produce undercarboxylated vitamin K-dependent clotting factors with reduced procoagulant activity. This prolongs the PT, chiefly due to depression in the levels of Factors II, VII and X. Successful oral anticoagulant therapy with coumarins requires careful monitoring of the patient's PT in order to achieve an effective level of anticoagulation while minimizing bleeding complications (reviewed by Hirsh et al. [1]).
The PT test is accomplished by mixing citrated plasma samples with a thromboplastin reagent and measuring the time to clot formation. An active ingredient in thromboplastin reagents is tissue factor, the protein responsible for triggering the blood clotting cascade through the extrinsic pathway [2]. Originally, thromboplastin reagents were prepared from relatively crude extracts of tissues (usually brain or placenta) of human or animal origin. More recently, however, newer generation thromboplastin reagents have been developed that are based on purified, recombinant human tissue factor that has been reconstituted into phospholipid vesicles [3,4]. Because thromboplastin reagents vary widely in composition and manner of preparation, their sensitivities in monitoring oral anticoagulant therapy also vary widely. More sensitive thromboplastin reagents exhibit a more marked prolongation in the patient's PT as a consequence of oral anticoagulant therapy than do less sensitive thromboplastin reagents. Left uncorrected, this can result in markedly different dosing of oral anticoagulants [1].
Several years ago, a system was introduced to correct for differences in the sensitivities of thromboplastin reagents to coumarin therapy. Termed the International Sensitivity Index (ISI), this scheme has gained wide acceptance for measuring the responsiveness of a particular thromboplastin reagent to the decrease in coagulation proteins induced by administration of coumarin drugs [1]. The process for assigning an ISI value to a given lot of thromboplastin reagent is begun by measuring the clotting times of a collection of plasma samples consisting of a relatively large group of normal donors and patients who are stably anticoagulated with coumarins [5]. The clotting times of these same plasmas are also measured using an international reference thromboplastin of known ISI value. The ISI value of the new thromboplastin reagent is then derived from the slope of the regression line fitted to the clotting times obtained with the two reagents, plotted on log-log graphs. These ISI values are ultimately traceable to an original international thromboplastin standard, which was assigned an ISI value of 1.00 [5]. The more sensitive a thromboplastin reagent is to the changes induced by coumarin therapy, the lower its ISI value. In general, thromboplastin reagents with ISI values near 1.0 have been the most desirable. Indeed, newer generation reagents (including those based on recombinant thromboplastin) have typically been manufactured to have ISI values near 1.0 [1].
The ISI value of a thromboplastin reagent is used to calculate the International Normalized Ratio (INR) for patient plasma samples. The INR is calculated by first dividing the patient's PT value by the mean PT value for 20 or more normal plasmas. This PT ratio is then raised to the ISI power, yielding the INR value, which in turn, is used by the treating physician to adjust the drug dose. The introduction of the INR reporting system has vastly improved the standardization of monitoring of oral anticoagulant therapy, and can be credited with decreasing bleeding complications for oral anticoagulant therapy [1].
Although the ISI/INR scheme has revolutionized the way in which oral anticoagulant therapy is monitored, it has been criticized on a number of grounds. For example, experience with this system has demonstrated that a single ISI value for each new lot of thromboplastin reagent is not sufficient. Instead, ISI values are determined for each combination of thromboplastin lot and the type of coagulometer used to measure clotting times [6]. For careful monitoring of patient INR values, some have recommended that clinical coagulation laboratories perform local calibrations using plasma calibrants for particular combinations of thromboplastin reagent and instrumentation used to measure patient PT values [6]. In addition, the determination of ISI depends upon a linear relationship (on log-log plots) between clotting times determined with the test and reference thromboplastin reagents, but a number of instances of deviation from linearity have been reported, especially for patients with high INR values [7–9]. Recombinant thromboplastin reagents have also been criticized as being overly sensitive to changes in Factor VII levels, relative to tissue-derived thromboplastin reagents [8, 10].